Compositions for Inducing Immune Response Comprising Inverted Microsomes

ABSTRACT

A vaccine composition is provided which comprises inverted microsomes or fragments thereof from an animal cell in association with an externally disposed peptide antigen and a protein of the MHC.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 10/566,823 filedJun. 20, 2006, which is the U.S. National Stage filing of InternationalApplication Serial No. PCT/GB2004/003285 filed Jul. 30, 2004, whichclaims priority to GB 0318096.5 filed Aug. 1, 2003, each of which isincorporated herein by reference in its entirety.

The present invention relates to a novel peptide-based vaccines, uses ofsuch vaccines in prophylactic and therapeutic treatment of human andanimal diseases, such as viral infection and cancer.

Most of the successful vaccines depend on neutralising antibodies raisedby classic attenuated or killed pathogens. However, for pathogenscausing chronic infection—such as HIV, hepatitis C virus, mycobacteriaand parasites—or in the case of cancer, a T-cell mediated immuneresponse is crucial. Molecular understanding of MHC antigen presentationand the T-cell immune responses led to the use of defined antigenicpeptide plus cytokines and/or co-stimulatory molecules in attempts todevelop vaccines. One of the basic problems in all these attempts wasthe difficulty to reconstitute an antigen delivery system that isqualitatively and quantitatively similar to antigen presenting cells(APC) in vivo.

CD8+ cytotoxic T lymphocytes (CTL) recognise antigens as small antigenicpeptides that assemble with major histocompatibility complex (MHC) classI molecules. The antigenic peptides are generated in the cytosol of APCand subsequently translocated into the lumen of the endoplasmicreticulum (ER) (Rock, K. L. & Goldberg, A. L. Annu Rev Immunol 17,739-779 (1999)). The MHC class I heavy chain is synthesised and insertedinto the lumen of the ER and where it forms a dimer withb2-microglobulin (b2M) (Natarajan et al Rev Immunogenet 1, 32-46 (1999);Pamer E, & Cresswell P, Annu Rev Immunol. 16 323-358 (1998)). The dimersare retained in the ER until they assemble with proper antigenicpeptides. The process of MHC class I dimer and assembly with peptides inthe ER is catalysed by chaperones such as BIP, calnexin, calreticulin,and Erp57 (Paulsson K, & Wang P., Biochim Biophys Acta. 1641(1) 1-12(2003)).

The assembled MHC class I are rapidly expressed on the cell surface ofAPC, such as infected or malignant cells. The recognition of peptide-MHCclass I by T cell receptor leads the CTL to kill target cells expressinginfectious or tumor antigens. Following the identification of CTLrecognized epitopes from viral or cancer proteins, syntheticpeptide-based vaccines designed to elicit T-cell immunity became anattractive approach to the prevention or treatment of infectious andmalignant diseases (Furman M H, & Ploegh H L., J Clin Invest. 110(7)875-9 (2002); Berinstein N. Semin Oncol. 30 (3) (Suppl 8), 1-8 (2003);Falk et al Nature 348, 248-251. (1990); (Van Bleek G M, & Nathenson SG., Nature 348: 213-216 (1990); Kast, W. M., & Melief, C. J. Immunol.Lett. 30:229-232 (1991)). There are a number of different forms ofpeptide vaccines based on these delivery systems. The simplest form ispeptides dissolved in aqueous solutions. Direct injection of solubleantigenic peptides was shown to be unsuccessful at stimulating CTLresponses, either because of their rapid biodegradation or induction ofT cell anergy resulting from the antigenic stimulation by immature APC(Kyburz, D. et al. Eur. J. Immunol. 23:1956-1962 (1993); Toes, R. E etal Proc. Natl. Acad. Sci. USA. 93:7855-7860 (1996); Amoscato et al J.Immunol. 161, 4023-4032 (1998)). An additional complication reportedfrom the use of synthetic peptide-derived vaccines is the induction ofCTLs that, while they are capable of killing target cells that areexogenously pulsed with peptide, they are not able to recognise targetcells that naturally process and present the peptide epitope, such asinfected or malignant cells (Dutoit, V. et al. J. Clin. Invest.110:1813-1822 (2002)).

It has been reported that MHC class I antigen presentation isqualitatively controlled in the ER for selecting correct peptides. Onlythe correctly assembled MHC class I could express on the surface of APC.The use of adjuvants did little to increase the presentation quality ofsynthetic peptides (Schijns, V. E. 2001. Crit. Rev. Immunol. 21:75-85(2001). An improved version of the peptide-vaccine has been constructedas an artificial lipo-membrane (BenMohamed et al Lancet Infect Dis.2(7), 425-31 (2002)) with peptide-loaded recombinant MHC class I.Although liposome strategy is able to incorporate peptide bound MHCclass I molecules in the lipid membrane before injection into patients,the sophisticated loading system in the ER of APC could not be easilyimitated by a simple mixture of recombinant MHC class I, syntheticpeptide and liposomes. Only a few peptides would assemble withrecombinant MHC class I in vitro (Ostergaard Pedersen L, et al Eur JImmunol. 31(10), 2986-96 (2001).

In addition, the incorrect orientation of inserted MHC class I and lackof co-stimulatory molecules made it difficult to induce effective immuneresponses. Since the professional APCs have the unique ability ofpresenting optimal antigen and for initiating a cellular immune responseby naïve T cells, strategies are being developed to generate autologousdendritic cells (DC), a key APC, as vaccine vehicles ex vivo(Banchereau, J. et al. Annu. Rev. Immunol. 18:767-811 (2000)). Initialstudies showed that antigenic peptide-pulsed DC used as vaccines in vivocould induce a CTL response (Tsai, V. et al J. Immunol. 158:1796-1802(1997)). Despite the positive evidence reported from a number of humanclinical trials, there is no biochemical evidence showing that thepulsed peptides are indeed loaded on the surface MHC class I, whichquestions the efficiency of peptide-pulsed APCs to induce effectiveimmune responses.

There is therefore a need for a vaccine preparation that can overcomethese problems and present a therapeutically effective alternative toconventional vaccines. Such vaccines should achieve the quality of theendogenous presented antigen by APC cells while preserving high efficacyand avoiding side effects.

According to a first aspect of the invention, there is provided avaccine composition comprising isolated inverted microsomes from ananimal cell, or membrane fragments thereof, in association with anexternally disposed peptide antigen and a protein of the MajorHistocompatibility Complex (MHC).

The microsomes of the present invention are derived from an animal celland may therefore arise from the following compartments present in aeukaryotic cell: endoplasmic reticulum, lysosome; endosome, orcomponents of the endocytic pathway.

The microsome may be isolated with a protein of the MHC already presentin the membrane of the microsome or of the fragment. Alternatively, theMHC protein can be introduced into the microsome or fragmentsubsequently. The ER derived microsomes contain both MHC class I andclass II molecules (Bryant et al Adv Immunol. 80, 71-114 (2002)).

The present invention is equally applicable with respect to the MHCclass I restricted antigenic peptides as well as the MHC class IImolecules. The protein of the MHC in the composition may be from aheterologous source with respect to the cell from which the microsomesare obtained.

The MHC family of proteins are encoded by the clustered genes of themajor histocompatibility complex (MHC). MHC molecules are expressed onthe cells of all higher vertebrates. They were first demonstrated inmice and called H-2 antigens (histocompatibility-2 antigens). In humansthey are called HLA antigens (human-leucocyte-associated antigens)because they were first demonstrated on leucocytes (white blood cells).Class I and class II MHC molecules are the most polymorphic proteinsknown—that is, they show the greatest genetic variability from oneindividual to another—and they play a crucial role in presenting foreignprotein antigens to cytotoxic and helper T cells, respectively. Whereasclass I molecules are expressed on almost all vertebrate cells, class IImolecules are restricted to a few cell types that interact with helper Tcells, such as B lymphocytes and macrophages. Both classes of MHCmolecules have immunoglobulin-like domains and a single peptide-bindinggroove, which binds small peptide fragments derived from foreignproteins. Each MHC molecule can bind a large and characteristic set ofpeptides, which are produced intracellularly by protein degradation.After they form inside the target cell, the peptide-MHC complexes aretransported to the cell surface, where they are recognized by T cellreceptors. In addition to their antigen-specific receptors thatrecognize peptide-MHC complexes on the surface of target cells, T cellsexpress CD4 or CD8 co-receptors, which recognize non-polymorphic regionsof MHC molecules on the target cell: helper cells express CD4, whichrecognizes class II MHC molecules, while cytotoxic T cells express CD8,which recognizes class I MHC molecules. (Alberts et al, “MolecularBiology of the Cell”, 3rd edition, 1229-1235 (1994)).

The MHC class I consists of heavy chain and Beta-2-microglobulin. HumanMHC class I heavy chains are encoded by three separate genetic locicalled HLA A, B, C. They are noncovalently associated with a smallprotein called beta-2-microglobulin. An example of a human MHC class Iprotein is HLA class I histocompatibility antigen, A-2 alpha chainprecursor (MHC class I antigen A*2) is shown in FIG. 13 (databaseaccession no. P01892); or HLA class I histocompatibility antigen, B-7alpha chain precursor (MHC class I antigen B*7) as shown in FIG. 13(database accession no. P01889).

MHC class II are composed of two noncovalently bonded chains an α-chainand an β-chain. chain. Both chains are coded by genes in I-regionassociated (Ia) antigens. Examples of such proteins are HLA class IIhistocompatibility antigen, DRB3-1 beta chain precursor (MHC class Iantigen DRB3*1) shown in FIG. 14 (database accession no. P79483); andMHC class II histocompatibility antigen HLA-DQ alpha 1 (DQw4specificity) precursor, also shown in FIG. 14 (database accessionA37044).

The sequences of the MHC class I and II cDNAs and genomic DNAs arepublished and available.

All eucaryotic cells have an endoplasmic reticulum (ER). Its membranetypically constitutes more than half of the total membrane of an averageanimal cell. It is organized into a netlike labyrinth of branchingtubules and flattened sacs extending throughout the cytosol. The tubulesand sacs are all thought to interconnect, so that the ER membrane formsa continuous sheet enclosing a single internal space. This highlyconvoluted space is called the ER lumen or the ER cisternal space, andit often occupies more than 10% of the total cell volume. The ERmembrane separates the ER lumen from the cytosol, and it mediates theselective transfer of molecules between these two compartments.

The ER plays a central part in lipid and protein biosynthesis. Itsmembrane is the site of production of all the transmembrane proteins andlipids for most of the cell's organelles, including the ER itself, theGolgi apparatus, lysosomes, endosomes, secretory vesicles, and theplasma membrane. The ER membrane also makes a major contribution tomitochondrial and peroxisomal membranes by producing most of theirlipids. In addition, almost all of the proteins that will be secreted tothe cell exterior—as well as those destined for the lumen of the ER,Golgi apparatus, or lysosomes—are initially delivered to the ER lumen(Alberts et al, “Molecular Biology of the Cell”, 3rd edition, 577-595(1994)).

The lysosome is a specialised organelle containing specialised enzymesfor the degradation of internal cellular proteins that are required tobe destroyed, or for the destruction of external foreign proteins orparasites that have been targeted for destruction by the immune system.

The endosome is a cell organelle that forms part of the endocyticpathway in the cell. There is a constant flow of endocytic vesicles thatflow from the cell surface to the endosome or to the lysosome. Thevesicles form by a process of “budding-off” from the external plasmamembrane, known as invagination, or the vesicles can form from theinternal cell organelles to which they ultimately return. Endocytosis isthe process by which a cell internalises external receptors with orwithout bound ligand and also one way by which the cell can sample itsexternal environment.

Compositions in accordance with the present invention may be optionallyformulated with an appropriate adjuvant, and/or cytokines that promoteT-cell responses, such as an interferon or an interleukin, e.g. IL-2,IL-15, IL-6, GM-CSF, IFNγ, other cytokines promoting T-cell responses,and/or conventional adjuvant. These can be suitably mixed with themicrosomes loaded with antigen prior to administration, or may besuitably prepared as membrane-bound constituents of the microsomes.

Microsomes in the context of the present invention are the cell freemembrane vesicles of the endoplasmic reticulum (ER), lysosomal, orendosomal compartments of any animal cell able to present antigenicpeptide by means of the Major Histocompatibility Complex (MHC). Thedefinition of ER-derived microsomes is based on the presence ofso-called “ER-markers” which are proteins normally resident in the ER,such as BIP, p58, calnexin, calreticulin, tapasin. The definition of alysosomal-derived microsome is based on the presence of the specificmarkers LAMP1 and/or LAMP2. Microsomes are recognised as such by theirmorphology as seen under the electron microscope following preparationfrom an animal cell.

The microsomes contained in a composition of the present invention canbe isolated by any convenient means. Suitable methods include those ofSaraste et al and/or Knipe et al (Saraste et al Proc. Natl. Acad. Sci.U.S.A. 83, 6425-6429 (1986) and Knipe et al J. Virol. 21, 1128-1139(1977)). Such methods comprise homogenisation of cells or tissues,followed by separation of the cell nucleus by centrifugation at 7500 rpmfor 10 minutes, then recovering the “rough” microsomes by centrifugationat 15500 rpm for 54 minutes. “Rough” microsomes are microsomes that haveribosomes attached. The resuspended “rough” microsomes are then furtherpurified by centrifugation through a sucrose cushion for differentialcentrifugation at 110,000 g for 60 minutes. The rough microsomes weresubfractionated by further centrifugation at 37,000 rpm for 10 hours ona sucrose gradient (to reach isopyknic conditions), and the ERcontaining fractions determined by Western blotting with appropriateantibody, for example anti-p58 antibody.

Inverted microsomes are the result of further processing, e.g. repeatedfreeze-thaw process steps, carried out on isolated microsomes whichcauses the disruption and reformation of the external membrane of themicrosome such that the “inside” face of the membrane is presented onthe “outside” of the inverted microsome. The microsomes that result fromsuch processing are therefore described as “inside-out” or “inverted”microsomes. The process of preparing the “inside-out” or invertedmicrosomes results in the absence of the lumen structure seen inordinary microsome preparations.

In compositions according to the present invention, the microsome maycomprise a membrane fragment thereof. Suitably, such membrane fragmentsmay be prepared by the method comprising the use of detergents orrepeated freeze-thawing or sonication to break the microsome structure.Such fragments may also be similarly loaded with peptide antigen to forma composition of the present invention. Preferably, the membranefragments are derived from intracellular membranes with markers specificto the ER or to the lysosomes.

In a vaccine composition of the present invention, there may also be apercentage of microsomes with a non-inverted structure, i.e. a membraneorientation that corresponds to the in situ arrangement after standardmicrosome preparation with an “inside” corresponding to the lumen of theER, endosome or lysosome prior to microsome preparation. However, atleast about 75% to about 95%, suitably at least about 90% of microsomesin the vaccine compositions of the invention have a reversed membraneorientation to in situ microsomes and are therefore described as being“inside-out” or inverted microsomes. The compositions may thereforeadditionally comprise a percentage of non-inverted microsomes.

In other embodiments of the invention, the composition may be morehomogenous, and so may comprise at least about 95%, 96%, 97%, 98%, 99%or 100% of microsomes having an inverted or reversed (or “inside-out”)membrane orientation compared to microsomes prepared from cells withoutfurther processing.

The microsomes may be loaded with antigen first and then subjected tofurther processing so as to provide inverted or “inside-out” microsomesthus exposing the inner surface of the ER membrane, or the microsomescan be prepared from a cell source where the preferred antigen peptideis already present in the microsomes, or the microsomes may be processedto provide inverted or “inside-out” microsomes first and thensubsequently loaded with antigen.

Lysosomes and endosomes can be prepared by an equivalent procedure.Lysosomal microsomes which are purified from the endocytotic compartmentof the animal cell include both lysosomes and endosomes. Afterfractionation of the total cellular membranes in the purificationprocedure for the preparation of ER-derived microsomes, the lysosomalmembranes are defined by antibodies to its markers LAMP1 and LAMP2.

The purified lysosomal microsomes are then processed to yield invertedor “inside-out” microsomes or membrane fragments as described abovewhich, if necessary, can then be loaded with MHC restricted peptidesunder acid conditions, such as for example at a pH of less then pH3,preferably from pH 3 to pH 3, suitably at around pH 2.5.

The animal cell from which the isolated microsome population is to beprepared can be any generally convenient cell type that has MHCmolecules expressed by the cell. For example, cells of the blood or ofthe immune system such as, B-cells and macrophages, the so-calledantigen presenting cells (APCs). However, cell types could also be usedfrom tissues such as liver, kidney, lung, brain, heart, skin, bonemarrow, pancreas etc.

The cells may be of a human or of a non-human animal. Suitably, theanimal is a mammal. The animal may be a rodent species, e.g. a mouse, arat or a guinea pig, or another species such as rabbit, or a canine orfeline, or an ungulate species such as ovine, porcine, equine, caprine,bovine, or a non-mammalian animal species, e.g. an avian (such aspoultry, e.g. chicken or turkey).

The cells from which the microsomes are prepared may be a cell line inculture. The cell line may be an immortalised cell line. The cell linemay be ultimately derived from a non-embryonic tissue source.

In certain embodiments of the invention, the source of cells may be agenetically modified source of animal cells, such as a cell line, or atransgenic non-human animal. The cells or tissue from which themicrosomes are prepared may be a humanised animal tissue or cell from atransgenic non-human animal whose genome has been modified by theinsertion of one or more human genes.

In embodiments of the invention relating to microsomes prepared from atransgenic non-human animal or transgenic cell line, the transgenesis isthe introduction of an additional gene or genes or protein-encodingnucleic acid sequence or sequences. The transgene may be a heterologousgene or an additional copy of a homologous gene, optionally under thecontrol of a constitutive promoter or an inducible promoter. Thetransgenesis may be transient or stable transfection of a cell or a cellline, or an episomal expression system in a cell or a cell line.

However, it is in the field of human medicine, in which the compositionsof this aspect of the invention are expected to find greatestapplication as vaccines. It is therefore preferred that the source ofcells from which the microsomes are prepared has an MHC allotype that iscompatible to the MHC of the recipient of the composition when used as avaccine.

In one embodiment according to this aspect of the invention, the sourceof cells from which the microsomes are prepared may be the ultimaterecipient of the composition when used as a vaccine.

Alternatively, a suitable source of human cells may be from a cell line,for example a non-embryo derived cell-line, suitably a B-cell line suchas cell line 221. Such cell lines may also advantageously not expressproteins of the Major Histocompatibility Complex (MHC) type class Iand/or class II. This embodiment of the invention may be a morepreferred embodiment for the manufacture of vaccines on a commercialscale, where non-individual vaccines are produced from such cell lineswhich have been engineered with different MHC allotypes.

Cell line 221 is an example of such a MHC negative cell line. Theabsence of a native MHC class I expression in such cells permits themodification of the cell line to express MHC class I of any desiredgenotype. This may be particularly important in achieving the fullimmunising effects of the vaccine composition, since different humanpopulations express different MHC proteins. In such compositions of theinvention, the MHC protein may therefore be of a heterologous sourcewith respect to the cell from which the microsomes are obtained.

Some of the MHC class I, like HLA A2, are expressed in more than 20% ofthe population. In circumstances where a MHC negative cell line is used,one or more than one compatible MHC gene is transfected into the cellline by means of conventional gene transfer methods and the transgene isconstructed into a expression vector. The expression cassette ofexpression construct normally includes standard promoter, such as CMVpromoter, or elongation factor I promoter or actin promoter, enhancer,inserted transgene and the poly-A signal to achieve optimal expression.Before transfection, the expression cassette will be isolated from theplasmid backbone to avoid the expression of bacterial plasmid genes intransfected cells. The sequences of the MHC class I and II cDNAs andgenomic DNAs are published and available. A MHC class I transfectantsBank can be constructed by using MHC class I negative or selected MHCclass positive cell lines to transfect most of the MHC class I genes,respectively. The selection of the expression cassette will be dependenton the optimal expression of the transgene.

Transfection of the antigen presenting cells may be achieved usingstandard recombinant techniques, e.g. using a suitable vector comprisinga nucleic acid sequence encoding a MHC protein of interest. The term“vector” generally refers to any nucleic acid vector which may be RNA,DNA or cDNA. The vector can be described alternatively as an “expressionvector”.

The terms “vector” or “expression vector” may include, among others,chromosomal, episomal, and virus-derived vectors, for example, vectorsderived from bacterial plasmids, from bacteriophage, from transposons,from yeast episomes, from insertion elements, from yeast chromosomalelements, from viruses such as baculoviruses, papova viruses, such asSV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabiesviruses and retroviruses, and vectors derived from combinations thereof,such as those derived from plasmid and bacteriophage genetic elements,such as cosmids and phagemids. Generally, any vector suitable tomaintain, propagate or express nucleic acid to express a polypeptide ina host may be used for expression in this regard. The vector may beconstructed from a bacterial plasmid, for example the bacterial plasmidpUC18.

The vector may provide for specific expression. Such specific expressionmay be inducible expression or expression only in certain types of cellsor both inducible and cell-specific. Preferred among inducible vectorsare vectors that can be induced for expression by environmental factorsthat are easy to manipulate, such as temperature, nutrient additives,hypoxia and/or the presence of cytokines or other biologically activefactors. Particularly preferred among inducible vectors are vectors thatcan be induced for expression by changes in the levels of chemicals, forexample, chemical additives such as antibiotics. A variety of vectorssuitable for use in the invention, including constitutive and inducibleexpression vectors for use in prokaryotic and eukaryotic hosts, are wellknown and employed routinely by those skilled in the art.

Recombinant expression vectors will include, for example, origins ofreplication, a promoter preferably derived from a highly expressed geneto direct transcription of a structural sequence, and a selectablemarker to permit isolation of vector containing cells after exposure tothe vector.

Mammalian expression vectors may comprise an origin of replication, asuitable promoter and enhancer, and also any necessary ribosome bindingsites, polyadenylation regions, splice donor and acceptor sites,transcriptional termination sequences, and 5′-flanking non-transcribedsequences that are necessary for expression. Preferred mammalianexpression vectors according to the present invention may be devoid ofenhancer elements.

The promoter sequence may be any suitable known promoter, for examplethe human cytomegalovirus (CMV) promoter, the CMV immediate earlypromoter, the HSV thymidine kinase promoter, the early and late SV40promoters or the promoters of retroviral LTR's, such as those of theRous sarcoma virus (“RSV”), and metallothionein promoters, such as themouse metallothionein-I promoter. The promoter may comprise the minimumsequence required for promoter activity (such as a TATA box withoutenhancer elements), for example, the minimal sequence of the CMVpromoter (mCMV). Preferably the promoter is a mammalian promoter thatcan function at a low basal level devoid of an enhancer element.

Preferably, the promoter is contiguous to the nucleic acid sequenceencoding the MHC protein to be transfected into the antigen presentingcell. It is contemplated that variants, for example, homologues ororthologues, of the promoters described herein are part of the presentinvention.

The backbone of the expression vector of the first aspect of theinvention may be derived from a vector devoid of its own promoter andenhancer elements, for example the plasmid vector pGL2. Enhancers areable to bind to promoter regions situated several thousands of basesaway through DNA folding (Rippe et al TIBS 1995; 20: 500-506 (1995)).

The expression vectors may also include selectable markers, such asantibiotic resistance, which enable the vectors to be propagated.

The nucleic acid sequences of the vector containing nucleic acidencoding the MHC protein to be transfected may encode a reporter proteinas described above, such as a chloramphenicol acetyl transferase (“CAT”)transcription unit, luciferase or green fluorescent protein (GFP). Theapplication of reporter genes relates to the phenotype of these geneswhich can be assayed in a transformed cell and which is used, forexample, to analyse the induction and/or repression of gene expression.Reporter genes for use in studies of gene regulation include other wellknown reporter genes including the lux gene encoding luciferase whichcan be assayed by a bioluminescence assay, the uidA gene encodingβ-glucuronidase which can be assayed by a histochemical test, the lacZgene encoding β-galactosidase which can be assayed by a histochemicaltest, the enhanced green fluorescent protein which can be detected by UVlight, UV microscopy or by FACS.

The DNA comprising the nucleic acid sequence of the MHC protein may besingle or double stranded. Single stranded DNA may be the coding orsense strand, or it may be the non-coding or anti-sense strand. Fortherapeutic use, the nucleic acid sequences are in a form capable ofbeing expressed in the subject to be treated.

The termination sequences in the vector may be a sequence of adenylatenucleotides which encode a polyadenylation signal. Typically, thepolyadenylation signal is recognisable in the subject to be treated,such as, for example, the corresponding sequences from viruses such as,for human treatment, the SV40 virus. Other termination signals are wellknown in the art and may be used.

Preferably, the polyadenylation signal is a bidirectional terminator ofRNA transcription. The termination signal may be the polyadenylationsignal of the simian 40 virus (SV40), for example the SV40 late poly(A).Alternatively, the termination sequence may be the polyadenylationsignal of bovine growth hormone which results in maximal expression whencombined with a CMV promoter (Yew et al. Human Gene Therapy, 8: 575-584(1997)).

In addition the expression vector may comprise a further polyadenylationsequence, for example an SV40 early poly(A). Such a further poly (A) maybe located upstream of the nucleic acid sequence encoding the MHCprotein to reduce cryptic transcription which may have initiated withinthe vector thereby ensuring that basal gene expression from the vectoris minimal.

Gene expression from integrated viral genomes may be susceptible tochromosomal positional effects. Such effects include transcriptionalsilencing and promoter activation by nearby heterologous enhancers. Inaddition, integrated sequences can activate expression of nearby genesand oncogenes. These effects are reduced through the use of elementswhich form boundaries to the inserted viral genome. Insulators aregenetic elements such as the chicken β-globin 5′ DNase I hypersensitivesite (5′HS4) which mark a boundary between an open chromatin domain anda region of constitutively condensed chromatin.

Other elements termed scaffold or matrix attachment regions (S/MAR)anchor chromatin to nuclear structures and form chromosomal loops whichmay have a physiological role in bringing distal regulatory elementsinto close proximity to a corresponding promoter. An example is locatedin the human interferon-γ locus and is termed the IFN-SAR. Bothinsulators and S/MAR can reduce position effects with greatest activitydemonstrated when they were combined in a lentiviral vector (Ramezani etal, Blood 101: 4717-24, (2003)). Clearly such elements can be of benefitin regulated vectors such as those described herein after they areintegrated into the host cell genome.

The compositions of the present invention comprise a microsome, or afragment thereof, in association with an externally disposed peptideantigen that has been loaded into the microsome. The association may besuch that the peptide antigen is inserted in the membrane of themicrosome such at least one epitope of the peptide antigen is exposedwith respect to the outer membrane of the microsome. The membrane ofmicrosomes further contains a protein of the MHC that presents thepeptide antigen to T-cells in order for the antigen to be recognised bythe immune system. The MHC protein is either naturally present in thecell organelles of the cell from which the microsomes were produced, orit is a MHC protein that has been transfected into the cell throughrecombinant DNA techniques and expressed, prior to preparation of themicrosomes. The inserted antigenic peptide and the MHC protein form anassociation in the membrane of the microsome which permits externaldisposition of the proteins for interaction with the cells of the immunesystem.

The antigenic peptides may be introduced or loaded into the microsome bymeans of incubating the microsome with the peptide antigen in thepresence of a nucleoside triphosphate (NTP), for example adenosinetriphosphate (ATP) and NTP re-generation system. It appears that an NTP,such as ATP, facilitates the incorporation of the peptide antigen intothe microsome through protein transporters located in the membrane ofthe microsome. Without wishing to be bound unnecessarily by theory, itappears that once the microsome is incubated with the peptide antigen inthe presence of an NTP that the antigen is able to associate with MHCclass I proteins already present in the membrane of the microsome.Alternatively, the antigenic peptides may also loaded into themicrosomes after inside-out processing and in this case, the NTP is notrequired.

The antigenic peptide present in association with the microsome suitablyhas one or more epitopes. An epitope is the smallest part of an antigenrecognisable by the combining site of an immunoglobulin and may belinear or discontinuous. Therefore, any type of MHC binding peptides,natural or synthesized or artificially modified, is included.

The antigenic peptides may be from a source that is foreign, i.e.non-self, or self, i.e. an autoantigen. Foreign antigenic peptides mayoriginate from virus, bacteria, yeast, fungi, protozoa, or othermicro-organism (i.e. an infectious agent), or of higher life forms suchas plants or animals. In some embodiments of the invention, the antigenmay be an auto-antigen, for example an antigen expressed by a neoplasticcell or cell of a cancer tumour, a normal self-protein (in the case ofan tolerising vaccine of the invention for an auto-immune disorder).

Where the antigen is from a neoplastic cell or cell of a cancer tumour,the cell may be from a melanoma, lung adenocarcinoma, colon cancer,breast cancer or leukemia cell. Auto-immune disorders include, but arenot limited to, Multiple Sclerosis (MS), Systemic Lupus Erythamatosus,Type-1 or Insulin-dependent Diabetes, Antiphospholipid Syndrome,Myasthenia Gravis, Myositis, Sjogren's Syndrome and Rheumatoidarthritis.

In some embodiments of the invention, it may be preferred to prepare thecomposition with an antigenic peptide of more than one type, orantigenic peptides having a sequence modified to increaseimmunogenicity. The cell may also be transfected prior to thepreparation of the microsomes with more than one type of MHC moleculeswhich may be useful in the case of recipients of the compositions whenused as vaccines who have more than one type of MHC allotype.

In a preferred embodiment of this aspect of the invention, there isprovided a composition as defined above in which the ratio of antigen toMHC molecule in the microsome is optimal for the induction of a specificimmunoresponse, for example in the range of from 0.1 to 1.5, preferablyof from 0.2 to 1.2 or 0.5 to 1.0, and most preferably from 0.2-0.5 to1.0. The amount of loaded antigenic peptides may be different accordingto the level of immune response induced.

Defined antigenic peptides of major diseases can be readily selectedfrom the scientific literature or identified by bioinformatic tools,(Renkvist et al Cancer Immunol Immunother 50, 3-15 (2001); Coulie et alImmunol Rev 188, 33-42 (2002); De Groot et al Vaccine 19 (31), 4385-95(2001)).

For example, the influenza virus derived peptides SIINFEKL andASNENMETM, or the peptide YLQLVFGIEV from melanoma cells.

Table 1 shows details of Class I HLA-restricted cancer/testis antigens;Table 2 shows Class I HLA-restricted melanocyte differentiationantigens; Table 3 shows Class I HLA-restricted widely expressedantigens; Table 4 shows Class I HLA-restricted tumor specific antigens;Table 5 shows Class II HLA-restricted antigens; Table 6 shows epitopesderived from fusion proteins; and Table 7 shows frequency of epitopesrecognised by a given HLA allele.

Further examples are shown in Table 8 of Hepatitis C virus (HCV)peptides from Anthony et al Clinical Immunol., vol. 103, pages 264-276(2002); in Table 9 of Human Immunodeficiency Virus-1 (HIV-1) from Kaulet al J. Clinical Invest., vol. 107, pages 1303-1310 (2001; in Table 10of Hepatitis C Virus (HCV) peptides from Koziel et al J. Virol., vol.67, pages 7522-7532 (1993); and in Table 11 of Hepatitis C Virus (HCV)from He et al PNAS USA, vol. 96, pages 5692-5697 (1999).

The antigenic peptide epitopes may be present as a monomer or asrepeated sequence of the epitope, such as dimer, trimer, tetramer, orhigher multiple, such as a pentamer, hexamer, heptamer, octamer, nonameror decamer. Fragments of the epitope sequences can be used, as well asoverlapping sequences that include the epitope sequence.

The term “peptide” includes both polypeptide and protein, unless thecontext specifies otherwise.

Such peptides include analogues, homologues, orthologues, isoforms,derivatives, fusion proteins and proteins with a similar structure orare a related polypeptide as herein defined.

The term “analogue” as used herein refers to a peptide that possesses asimilar or identical function as a protein sequence described herein butneed not necessarily comprise an amino acid sequence that is similar oridentical to such an amino acid sequence, or possess a structure that issimilar or identical to that of a protein described herein. An aminoacid sequence of a peptide is “similar” to that of a peptide describedherein if it satisfies at least one of the following criteria: (a) thepeptide has an amino acid sequence that is at least 30% (morepreferably, at least 35%, at least 40%, at least 45%, at least 50%, atleast 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95% or at least 99%)identical to the amino acid sequence of a peptide described herein; (b)the peptide is encoded by a nucleotide sequence that hybridizes understringent conditions to a nucleotide sequence encoding at least 5 aminoacid residues (more preferably, at least 10 amino acid residues, atleast 15 amino acid residues, at least 20 amino acid residues, at least25 amino acid residues, at least 40 amino acid residues, at least 50amino acid residues, at least 60 amino residues, at least 70 amino acidresidues, at least 80 amino acid residues, at least 90 amino acidresidues, at least 100 amino acid residues, at least 125 amino acidresidues, or at least 150 amino acid residues) of a peptide sequencedescribed herein; or (c) the peptide is encoded by a nucleotide sequencethat is at least 30% (more preferably, at least 35%, at least 40%, atleast 45%, at least 50%, at least 55%, at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95% or at least 99%) identical to the nucleotide sequence encodinga peptide described herein.

Stringent conditions of hybridisation may be characterised by low saltconcentrations or high temperature conditions. For example, highlystringent conditions can be defined as being hybridisation to DNA boundto a solid support in 0.5M NaHPO₄, 7% sodium dodecyl sulfate (SDS), 1 mMEDTA at 65° C., and washing in 0.1×SSC/0.1%SDS at 68° C. (Ausubel et aleds. “Current Protocols in Molecular Biology” 1, page 2.10.3, publishedby Green Publishing Associates, Inc. and John Wiley & Sons, Inc., NewYork, (1989)). In some circumstances less stringent conditions may berequired. As used in the present application, moderately stringentconditions can be defined as comprising washing in 0.2×SSC/0.1%SDS at42° C. (Ausubel et al (1989) supra). Hybridisation can also be made morestringent by the addition of increasing amounts of formamide todestabilise the hybrid nucleic acid duplex. Thus particularhybridisation conditions can readily be manipulated, and will generallybe selected according to the desired results. In general, convenienthybridisation temperatures in the presence of 50% formamide are 42° C.for a probe which is 95 to 100% homologous to the target DNA, 37° C. for90 to 95% homology, and 32° C. for 70 to 90% homology.

A peptide with “similar structure” to that of a peptide described hereinrefers to a peptide that has a similar secondary, tertiary or quaternarystructure as that of a peptide described herein. The structure of apeptide can determined by methods known to those skilled in the art,including but not limited to, X-ray crystallography, nuclear magneticresonance, and crystallographic electron microscopy.

The term “fusion protein” as used herein refers to a peptide thatcomprises (i) an amino acid sequence of a peptide described herein, afragment thereof, a related peptide or a fragment thereof and (ii) anamino acid sequence of a heterologous peptide (i.e., not a peptidesequence described herein).

The term “homologue” as used herein refers to a peptide that comprisesan amino acid sequence similar to that of a peptide described herein butdoes not necessarily possess a similar or identical function.

The term “orthologue” as used herein refers to a non-human peptide that(i) comprises an amino acid sequence similar to that of a peptidedescribed herein and (ii) possesses a similar or identical function.

The term “related peptide” as used herein refers to a homologue, ananalogue, an isoform of, an orthologue, or any combination thereof of apeptide described herein.

The term “derivative” as used herein refers to a peptide that comprisesan amino acid sequence of a peptide described herein which has beenaltered by the introduction of amino acid residue substitutions,deletions or additions. The derivative peptide possess a similar oridentical function as peptides described herein.

The term “fragment” as used herein refers to a peptide comprising anamino acid sequence of at least 5 amino acid residues (preferably, atleast 10 amino acid residues, at least 15 amino acid residues, at least20 amino acid residues, at least 25 amino acid residues, at least 40amino acid residues, at least 50 amino acid residues, at least 60 aminoresidues, at least 70 amino acid residues, at least 80 amino acidresidues, at least 90 amino acid residues, at least 100 amino acidresidues) of the amino acid sequence of a peptide as described herein,mutatis mutandis. The fragment of may or may not possess a functionalactivity of such peptides.

The term “isoform” as used herein refers to variants of a peptide thatare encoded by the same gene, but that differ in their isoelectric point(pI) or molecular weight (MW), or both. Such isoforms can differ intheir amino acid composition (e.g. as a result of alternative splicingor limited proteolysis) and in addition, or in the alternative, mayarise from differential post-translational modification (e.g.,glycosylation, acylation, phosphorylation). As used herein, the term“isoform” also refers to a peptide that exists in only a single form,i.e., it is not expressed as several variants.

The percent identity of two amino acid sequences or of two nucleic acidsequences is determined by aligning the sequences for optimal comparisonpurposes (e.g., gaps can be introduced in the first sequence for bestalignment with the sequence) and comparing the amino acid residues ornucleotides at corresponding positions. The “best alignment” is analignment of two sequences which results in the highest percentidentity. The percent identity is determined by the number of identicalamino acid residues or nucleotides in the sequences being compared(i.e., % identity=# of identical positions/total # of positions×100).

The determination of percent identity between two sequences can beaccomplished using a mathematical algorithm known to those of skill inthe art. An example of a mathematical algorithm for comparing twosequences is the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci.USA (1990) 87:2264-2268, modified as in Karlin and Altschul (1993) Proc.Natl. Acad. Sci. USA 90:5873-5877. The NBLAST and XBLAST programs ofAltschul et al, J. Mol. Biol. (1990) 215:403-410 have incorporated suchan algorithm. BLAST nucleotide searches can be performed with the NBLASTprogram, score=100, wordlength=12 to obtain nucleotide sequenceshomologous to a nucleic acid molecules encoding a peptide sequence asdescribed herein. BLAST protein searches can be performed with theXBLAST program, score=50, wordlength=3 to obtain amino acid sequenceshomologous to a peptide as described herein. To obtain gapped alignmentsfor comparison purposes, Gapped BLAST can be utilised as described inAltschul et al, Nucleic Acids Res. (1997) 25:3389-3402. Alternatively,PSI-Blast can be used to perform an iterated search which detectsdistant relationships between molecules (Id.). When utilising BLAST,Gapped BLAST, and PSI-Blast programs, the default parameters of therespective programs (e.g., XBLAST and NBLAST) can be used.

Another example of a mathematical algorithm utilised for the comparisonof sequences is the algorithm of Myers and Miller, CABIOS (1989). TheALIGN program (version 2.0) which is part of the GCG sequence alignmentsoftware package has incorporated such an algorithm. Other algorithmsfor sequence analysis known in the art include ADVANCE and ADAM asdescribed in Torellis and Robotti Comput. Appl. Biosci. (1994) 10:3-5;and FASTA described in Pearson and Lipman Proc. Natl. Acad. Sci. USA(1988) 85:2444-8. Within FASTA, ktup is a control option that sets thesensitivity and speed of the search.

The skilled person is aware that various amino acids have similarproperties. One or more such amino acids of a substance can often besubstituted by one or more other such amino acids without eliminating adesired activity of that substance. Thus the amino acids glycine,alanine, valine, leucine and isoleucine can often be substituted for oneanother (amino acids having aliphatic side chains). Of these possiblesubstitutions it is preferred that glycine and alanine are used tosubstitute for one another (since they have relatively short sidechains) and that valine, leucine and isoleucine are used to substitutefor one another (since they have larger aliphatic side chains which arehydrophobic). Other amino acids which can often be substituted for oneanother include: phenylalanine, tyrosine and tryptophan (amino acidshaving aromatic side chains); lysine, arginine and histidine (aminoacids having basic side chains); aspartate and glutamate (amino acidshaving acidic side chains); asparagine and glutamine (amino acids havingamide side chains); and cysteine and methionine (amino acids havingsulphur containing side chains). Substitutions of this nature are oftenreferred to as “conservative” or “semi-conservative” amino acidsubstitutions.

Amino acid deletions or insertions may also be made relative to theamino acid sequence of a peptide sequence as described herein. Thus, forexample, amino acids which do not have a substantial effect on theactivity of such peptides, or at least which do not eliminate suchactivity, may be deleted. Amino acid insertions relative to the sequenceof peptides as described herein can also be made. This may be done toalter the properties of a protein of the present invention (e.g. toassist in identification, purification or expression, where the proteinis obtained from a recombinant source, including a fusion protein. Suchamino acid changes relative to the sequence of a peptide from arecombinant source can be made using any suitable technique e.g. byusing site-directed mutagenesis. The molecule may, of course, beprepared by standard chemical synthetic techniques, e.g. solid phasepeptide synthesis, or by available biochemical techniques.

It should be appreciated that amino acid substitutions or insertionswithin the scope of the present invention can be made using naturallyoccurring or non-naturally occurring amino acids. Whether or not naturalor synthetic amino acids are used, it is preferred that only L-aminoacids are present.

According to the present invention, purified microsomes representing theendoplasmic reticulum, lysosomes or endosomes in antigen-presentingcells (APC) can be used to load antigenic peptides on its MHC class I orII molecules. Results from in vitro and in vivo immunisation describedherein show that peptide-loaded microsome elucidates much strongerresponses than peptide-loaded APC measured by T cell proliferation andproduction of IL-2. By quantitating the amount of peptide-receptive MHCclass I molecules, the receptive class I molecules on APC surface arebelow the radio-chemical detection limit. However, a significant amountof peptide bound MHC class I is detected in the microsome. In addition,a similar amount of co-stimulatory molecules, B7.1 and B7.2 is detectedin microsomes in comparison to cell surface. Thus, the microsomes loadedwith antigenic peptides represent an effective vaccine composition.

The present invention has found that more than 50% of the MHC class Imolecules in the ER of APC are peptide receptive. By the process of“inside-out”, the microsomes loaded with Kb specific OVA-peptide caninduce T cell responses in vitro and in vivo. In contrast, the APCspulsed with same peptide have much less ability to stimulate T cellresponses. Given that the microsomes contain co-stimulatory molecules,the microsomes isolated from APCs represent promising vehicles forpeptide vaccines in the future for a wide variety of diseases.

In addition to transfecting selected MHC genes into the animal cellprior to microsome preparation, it may also be desirable to transfect orco-transfect the cells with genes encoding co-stimulatory molecules suchas B7 and/or the genes encoding cytokines, for example an interleukin oran interferon, such as IL-2. In the case of cytokines, the transgenewill be fused with trans-membrane domain of CD2 or CD4 for targeting thecytokines into the ER membrane. In addition, in order to enrich thelevel of MHC, co-stimulatory molecules and membrane-bound cytokines inthe ER, KDEL or other ER retention signalling (Nilsson T, & Warren G.,Curr Opin Cell Biol. 6 (4), 517-21 (1994)) will be tagged at theC-terminus of the transgenes for the retention of transgene products inthe ER. The expression cassettes for these transgenes are similar to MHCclass I transgenes.

According to the present invention, therefore, the vaccine compositionsmay be co-administered with one or more cytokines, such as an interferonor an interleukin, that can promote T cell immune response such as IL-2,IL-15, IL-6, GM-CSF, IFNγ, other cytokines promoting T cell responses,and/or conventional adjuvant. These can be suitably mixed with themicrosomes loaded with antigen prior to administration, or may besuitably prepared as membrane-bound constituents of the microsomes. Suchmembrane-bound substituents may be introduced using recombinant DNAtechniques, as discussed above, to engineer expression of the cytokinein the cell organelle that will ultimately be used to form themicrosomes, or alternatively the cytokines may be loaded into themicrosome membrane or bound to surface proteins.

A membrane bound cytokine expressed in a microsome preparation may beprepared by transfecting an antigen presenting cell with a constructcomprising a cytokine molecule fused to a membrane anchor protein,optionally with an ER-retention signal. For example, a microsomeincluding membrane bound IL-2 molecules can be prepared by constructinga vector comprising nucleic acid encoding a fusion protein comprisingthe CD2 membrane domain fused to the C-terminus of IL-2 and anER-retention signal, such as the 16 amino-acid sequence from E15-9Kadenovirus protein, where the ER-retention signal is fused to theC-terminus of the CD2 protein.

Expression of the vector in the antigen presenting cell leads toaccumulation of the cytokine in the organelles of the cell, i.e. the ER,which enables preparation of microsomes containing membrane-boundcytokine.

In addition, it may be convenient to include detection and monitoring ofthe specific immune responses towards the vaccine, for example bytechniques such as ELISA for detection of serum cytokine, e.g. IL-2and/or IFNγ, or an in vitro T cell response assay with peptide loadedmicrosomes, or a proliferative cell assay.

In its simplest form, the present invention provides a compositioncomprising an isolated microsome of the endoplasmic reticulum of ananimal cell, or a membrane fragment thereof, in association with anexternally disposed peptide antigen and a protein of the MajorHistocompatibility Complex (MHC). Suitably formulated for administrationas a vaccine.

According to a second aspect of the invention, there is provided acomposition according to the first or second aspects of the inventionfor use in medicine. This aspect of the invention therefore extends to amethod of treatment or prophylaxis of a subject suffering from a diseaseor condition, comprising the step of administering to the subject avaccine as defined above.

According to a third aspect of the invention, there is provided the useof a composition as defined above in the preparation of a vaccine forthe prophylaxis or treatment of a disease condition. The disease may bean infection caused by a micro-organism or virus, or it may be a cancerwhich is characterised by neoplastic cell growth and/or tumourformation. Alternatively, the disease may be an autoimmune condition,where a vaccine may have therapeutic use in inducing tolerance toself-antigens. Uses in accordance with this aspect of the invention alsoextend to methods of treatment of such disease conditions comprisingadministering said compositions to a subject in need thereof. Suitably,vaccine compositions of the present invention can be administered by anyconvenient route such as intramuscular, intravenous, intraperitoneal,oral or by injection in to the cerebrospinal fluid.

Diseases or conditions that can be treated using a vaccine of thepresent invention include, but are not limited to melanoma, lungadenocarcinoma, colon cancer, breast cancer or leukemia. Auto-immunedisorders include, but are not limited to, Multiple Sclerosis (MS),Systemic Lupus Erythamatosus, Type-1 or Insulin-dependent Diabetes,Antiphospholipid Syndrome, Myasthenia Gravis, Myositis, Sjogren'sSyndrome and Rheumatoid arthritis. In addition, viral infection, such asHIV infection, herpes virus infection, hepatitis C virus infection, orparasite infections, such as protozoan parasite infection of Plasmodium,the causative agent responsible for malaria, for example Plasmodiumfalciparum, Plasmodium vivax, Plasmodium berghei, Plasmodium yoelii orPlasmodium knowlesi, or another parasite such as Toxoplasma gondii, orTrypanosoma brucei, or Entamoeba histolytica, or Giardia lambia, orbacterial infection, such as E. coli 0157, Vibrio cholerae, etc.

It is also envisaged that the microsomes of the present invention whenloaded with peptide antigen may be fused with antigen presenting cells(APC) prior to administration of the combined preparation to thepatient. Suitably, the APC are taken from the patient prior totreatment, but may also be taken from an allogenic source. In suchcases, where an allogenic source of cells is used, immunosuppresivedrugs may also form part of the treatment protocol.

According to a fourth aspect of the invention, there is provided aprocess for the preparation of a vaccine composition as defined above,the process comprising incubating a population of microsomes and anantigenic peptide in the presence of a nucleoside triphosphate (NTP),followed by further processing to prepare inverted microsomes andformulating the resulting preparation in an physiological diluent andoptionally an adjuvant. Reagents such as glucose have ability topreserve the conformation of prepared microsome vaccine and may beincluded. Suitably, the incubated microsomes will be washed andresuspended in a vaccine solution of an physiological diluent containingan amount of antigenic peptides for preventing the dissociation of theMHC-peptide complex present in the microsome membrane.

The process of loading the microsomes with antigen is carried out in thepresence of a nucleoside triphosphate (NTP), such as ATP, GTP, CTP, TTP,or UTP. The antigen loading process may also be carried out in thepresence of more than one type of antigenic peptide. In this way aplurality of antigens can be loaded into the microsome.

Inverted microsomes may be prepared by further processing of themicrosomes that disrupts the membranes of the microsomes underconditions which allow the membrane to reform and which encourage theformation of “inside-out” microsomes. The use of repeated freeze-thawsteps are therefore suitable in this regard. For example, the microsomescan be suspended in a suitable medium, or buffer, e.g. Phosphatebuffered saline (PBS), and then briefly immersed into liquid nitrogenfor a suitable period of time, suitably from one to five minutes,preferably for two minutes, and then moved to 37° C., such as in awater-bath, for a suitable period of time, suitably for two to sixminutes, preferably for four minutes. The number of repeated steps offreeze-thaw may be from three to five, suitably four repeat steps.

In an alternative embodiment of this aspect of the invention, theprocess may comprise the loading of inverted microsomes, prepared asdescribed above, with antigen. For loading antigenic peptides intoinverted microsomes, the presence of an NTP may not be necessary(although it may be desirable).

According to a fifth aspect of the invention, there is provided a kit ofparts comprising a composition as defined above and one or more cytokineand/or adjuvant in sealed containers. Suitably, the kit will compriseinstructions for use in a method or use of the invention as definedabove.

According to a sixth aspect of the invention, there is provided a kit ofparts comprising a composition as defined above and one or more cytokineand/or adjuvant molecules for separate, subsequent or simultaneousadministration to a subject.

Preferred features for the second and subsequent aspects of theinvention are as for the first aspect mutatis mutandis.

In a particularly preferred embodiment of the invention there isprovided a vaccine composition for the prophylaxis or treatment of adisease that can be characterised by the expression of a defined antigenor a peptide sequence which is potentially immunogenic by an infectiousagent or which is characterised by the expression of an antigen of anative cell, in which the composition is prepared by:

-   -   (1) obtaining a sample of antigen presenting cells which express        MHC proteins;    -   (2) homogenising the cells under conditions such that a        preparation of microsomes is isolated;    -   (3) preparation of antigenic peptides, for example by means of        recombinant DNA technology, or from isolation from a natural        tissue source, or source of infectious agent, or in most cases        synthesised antigenic peptides.    -   (4) incubation of antigenic peptides and microsomes in the        presence of an NTP to load the microsomes with antigenic        peptides;    -   (5) further processing of microsomes loaded in step (4) to        prepare a population of inverted microsomes    -   (6) formulation of loaded inverted microsomes a vaccine in a        physiological diluent and/or adjuvant as appropriate

As described above, the microsomes may also be prepared from an isolatedpopulation of cells or a cell line. The cells or cell line may be havebeen transfected with a nucleic acid construct to express a protein ofchoice prior to microsome preparation.

According to the above protocol, the microsomes are loaded withantigenic peptide and then subsequently processed to prepare invertedmicrosomes. However, in an alternative embodiment, the invertedmicrosomes may be prepared first and then loaded with antigen, in whichcase the presence on an NTP in step (4) may not be required.

As discussed above, the vaccine compositions of the invention preferablycomprise inverted microsomes, more preferably a homogenous population ofinverted microsomes. However, non-inverted microsomes may also bepresent in the population of inverted microsomes.

The cells may be MHC negative so as to permit transfection of the cellline with appropriate nucleic acid encoding the MHC class molecule ofchoice for the vaccine. Preparation of antigen may also includesynthesis of antigenic peptides by means of chemical means.

The loading of non-inverted microsomes with antigenic peptide takesplace in the presence of an NTP. The loading of inverted microsomes doesnot appear to require the presence of an NTP, although it may bepreferred.

The invention will now be further described by way of reference to thefollowing Examples and Figures which are provided for the purposes ofillustration only and are not to be construed as being limiting on theinvention. Reference is made to a number of Figures in which:

FIG. 1 shows crosslinking of H2-Kb molecules by crosslinker-modified OVApeptide in the microsomes of RAW309Cr.1 cells. The ¹²⁵I-labeledANB-NOS-OVA peptide was mixed with the microsomes of RAW309Cr.1 cells inthe presence or absence of ATP-regenerating system or of nativeOVA-peptide at a ten-fold molar excess. The crosslinked H-2Kb wasindicated.

FIG. 2 shows concentration of OVA-peptide receptive H-2Kb in themicrosomes of RAW309Cr.1 cells. For semi-quantitation of OVA-peptidereceptive H-2Kb, 10 nMs of labelled peptide was incubated with themicrosomes or RAW309Cr.1 cells, respectively, under the UV irradiation.The H-2 molecules were precipitated by R218 antiserum and crosslinked Kbmolecules were quantitated by phospho-imaging.

FIG. 3 shows detection of H-2 molecules in the microsomes or on thesurface of RAW309Cr.1 cells. 30 μg proteins from NP40 lysates ofRAW309Cr.1 microsomes or RAW309Cr.1 cells were separated on 10%SDS-PAGE. The lysates were diluted at the titration indicated andseparated on the SDS-PAGE. Immunoblotting of H-2 molecules was detectedby R218 anti-H-2 antiserum.

FIG. 4 shows detection of B7.1, B7.2 and ICAM-1 in the microsomes ofRAW309Cr.1 cells. 30 μg proteins from NP40 lysates of RAW309Cr.1microsomes or RAW309Cr.1 cells were separated on 10% SDS-PAGE.Immunoblotting of B7. 1, B7.2 and ICAM-1 was detected by specificantibodies.

FIG. 5 shows stimulation of B3Z T cells by OVA-peptide editedmicrosomes. Microsomes from 2×10⁵ RAW309Cr.1 cells were used to load OVAor Ld-specific peptide as described in Material and Methods. 2×10⁵RAW309Cr.1 cells were pulsed with OVA peptide (see Material andMethods). After washing, peptide-pulsed 2×10⁵ RAW309Cr.1 cells,OVA-loaded microsomes, Ld-peptide loaded microsomes, and the microsomeswithout peptide were co-cultured with 10⁵ B3Z cells for over night. A)After washing with PBS, LacZ activity in B3Z cells was assayed by totalcellular lysates with the LacZ substrate ONPG. The absorbance (415 nM)was read after incubation for four hours at 37° C. B3Z cells culturedwith 100 nM OVA peptide and normal medium were used as positive andnegative control for the B3Z stimulation. B) The supernatants of thesecultures were submitted for measuring IL-2 production by ELISA. Theexperiment was repeated four times with similar results. Error barsindicate the SEM of triplicate cultures.

FIG. 6 shows stimulation of B3Z T cells by the microsomes of 2×10⁵RAW309Cr.1 cells pre-loaded with different concentrations ofOVA-peptides. Microsomes loaded with OVA-peptide at differentconcentration indicated were co-cultured with B3Z cells overnight beforethe assay of LacZ activity.

FIG. 7 shows OVA-peptide edited microsomes stimulates specific T cellresponses in vivo. C57BL/6 (H-2b) mice were primed i.s. by OVA-editedmicrosomes or Ld-peptide loaded microsomes or OVA peptide or OVA-pulsedRAW309Cr.1 cells and challenged by same stimulus after seven days. Sixdays after the challenge, enriched T cells were isolated from spleensand cultured at 10⁵ cells/well with stimulus indicated. The RAW309Cr.1cells were irradiated before co-culture with T cells. After three days,supernatants were harvested for cytokine ELISA (b) and cultures pulsedwith [³H]thymidine (a). The results are representative of groups of atleast three mice per treatment group and the experiment was repeatedfour times with similar results. Error bars indicate the SEM oftriplicate cultures. Similar set of experiments performed in Balb/c(H-2d) mice served as negative control.

FIG. 8 shows activation of TCR induced MAK kinases. 10⁷ T cells fromOVA-microsomes immunised C57BL/6 were stimulated with OVA-pulsedRAW309Cr.1, OVA-microsomes and anti-CD3/CD28, respectively. Activationof ERK and JNK was detected by anti-p-ERK and anti-p-JNK antibodies.Similar levels of ERK and JNK detected by anti-ERK and anti-JNK severedas loading control.

FIG. 9 shows OVA-receptive H-2Kb detected in microsomes, but not on thesurface of RAW309Cr.1 cells. The ¹²⁵I-labeled ANB-NOS-OVA peptide 10 nMwas mixed without or with native OVA peptide at concentrationsindicated. The mixed peptides were incubated with microsomes equivalentto 10⁷ RAW309Cr.1 cells or with 10⁷ RAW309Cr.1 cells. The crosslinkedH2-Kb molecules were indicated.

FIG. 10 shows the results of a study in which influenza viral peptideedited Kb-microsomes induced T-cell responses in vivo (five mice in eachgroup).

FIG. 11 shows electronmicrograph pictures of DC and prepared microsomes:(A) magnification ×12000 and (B) magnification ×40000 are preparedmicrosomes from RAW cells; (C) magnification ×40000 are microsomesinverted by repeated freeze-thaw and loaded with peptides, showing openor inverted microsomes. The loaded peptides can not be seen in thepicture.

FIG. 12 shows the results of a study carried out on MAGE-A2 specificT-cells from A2 melanoma patients.

FIG. 13 shows the amino acid sequences for the MHC class I antigens A2alpha chain precursor and B7 alpha chain precursor (Accession nos.P01892 and P01889, respectively).

FIG. 14 shows the amino acid sequences for the MHC class II antigensDRB3-1 beta-chain precursor and HLA-DQ alpha 1 (DQw4 specificity)precursor—human (Accession nos. P79483 and A37044, respectively).

MATERIAL AND METHODS Cell Lines and Animals

B3Z is a CD8 T cell hybridoma that expresses LacZ in response toactivation of T cell receptors specific for the SIINFEKL peptidepresented by H-2Kb MHC class I molecules. RAW309Cr.1, a Kd/Kb murinemacrophage cell line, used as APCs, was obtained from ATCC (ATCCTIB-69). All cells were cultured in Dulbecco's modified Eagle's mediumwith 10% fetal calf serum. Female C57BL/6 mice H-2b and Balb/c mice H-2dwere obtained at 6 weeks of age. All procedures with animals werecarried out in accordance with approved protocols.

Antibodies, Peptides, and Peptide Modification

All peptides were synthesised in a peptide synthesiser (model 431A,Applied Biosystems, Foster City, Calif.), using conventional F-mocchemistry, and were subsequently purified by HPLC. The purified peptideswere dissolved in PBS.

Peptide OVA 257-264 (SIINFEKL) was modified by substitution of thirdresidue isoleucine to tyrosine in order for iodination and by covalentlycoupling a phenylazide with a nitro group on the ε-amino group of lysineat position seven. This nitro group can be photoactivated. Thecrosslinker modification was performed by mixing 0.5 mg of ANB-NOS(N-5-azido-2-nitrobenzoyloxysuccinimide) in 200 μl DMSO with 100 μgpeptide in 100 μl PBS and 50 μl CPAS(3-[cyclohexylamino]-1-propanesulfonic acid) (0.5 M, pH 10). Thereaction was allowed to proceed for 30 min on ice. To remove the excessANB-NOS and ions, the mixture was purified by gel filtration on SephadexG-10 and subsequently by HPLC. An aliquot (1 μg) of the peptide waslabelled by chloramines-T-catalyzed iodination (¹²⁵I). The modificationand labelling experiments were performed in the dark.

Antisera, Immunoprecipitation, and SDS-PAGE

Rabbit antiserum to H2 (R218) was kindly provided by Dr. Sune Kvist,Karolinska Institute. Monoclonal antibody specific to confirmed H2 (Y3)was kindly provided by Tim Elliot, Cambridge University. Antisera toJNK, ERK, p-ERK and p-JNK were obtained from (Santa Cruz Biotechnology).Immunoprecipitation, immunoblotting and SDS-PAGE were performed asdescribed in Li et al (J Biol Chem. 274 (13), 8649-54 (1999)).Protein-A-Sepharose was obtained from Pharmacia (Uppsala, Sweden).

EXAMPLE 1 Preparation of Microsomes and Peptide Binding Assay

Microsomes from RAW309Cr.1, a Kd/Kb murine macrophage cell line wereprepared and purified according to the procedure of Saraste et al (Proc.Natl. Acad. Sci. U.S.A. 83, 6425-6429 (1986)). The immunogenetics ofclass I is Kb in RAW cells and Balb/c mice.

Preparation of microsomes from B cells based on a modification ofSaraste et al (Proc. Natl. Acad. Sci. U.S.A. 83, 6425-6429 (1986)) andKnipe et al (J. Virol. 21, 1128-1139 (1977)) for fractionation ofmicrosomal membranes was used. All steps were performed at 0-4° C.).

-   -   3×10⁹ cells are collected and washed once with cold PBS.    -   Resuspend the cells in 20 ml STKMM-buffer with 10 μl of PMSF        (100 mM).    -   Spin at 1500 rpm for 5 min at 4° C.    -   Resuspend in 10 ml H₂O (with 5 μl PMSF).    -   Homogenise in 40 ml Dounce, 20 strokes.    -   Add 30 ml STKMM and mixing well.    -   Pour over in JA-18 tubes.    -   Centrifuge at 7500 rpm for 10 min at 4° C.    -   Carefully collect supernatant to the new tubes.    -   Centrifuge at 15500 rpm for 54 min at 4° C.    -   Carefully wash the pellet with 10 ml STKMM buffer, then        resuspend the pellet in 1 ml RM buffer with a pipette and        homogenise in 15 ml douncer. The rough microsomes will be        diluted at a concentration of A_(OD280)=60.    -   Total microsomes (described above) were layered on top of 5 ml        of 0.33 M sucrose containing 5 mM benzamidine, layered in turn        on top of a sucrose cushion consisting of 1 ml of 2 M sucrose/5        mM benzamidine.    -   Centrifugation in an SW41 rotor for 60 min at 110,000×g yielded        a total microsome band on top of the cushion. The total        microsome band was carefully collected. Then, 2 M sucrose/5 mM        benzamidine was slowly added to the microsomes to give a final        concentration of 45% (w/v) sucrose.    -   Microsomes were subfractionated by flotation using a        modification of the method described in Paulsson et al (J Biol        Chem. 277 (21), 18266-71 (2002)). 100 μl of the total microsomes        in 3 ml of 45% (w/v) sucrose was placed at the bottom of an SW41        ultracentrifuge tube and overlaid with the following sucrose        solutions: 1 ml of 30% and 1.9 ml each of 27.5%, 25%, 22.5%, and        20.0% (all solutions contained 5 mM benzamidine).    -   After centrifugation at 4° C. for 10 h at 37,000 rpm (to reach        isopyknic conditions), 25 fractions of 300 μl each were        collected by upward displacement.    -   The ER fractions will be determined by western blotting with        anti-p58 antibody. (p58 is a ER protein).    -   The poured ER fractions will be used in peptide-loading and        immunisation experiments.

The cross-link mixture contained 50 or 100 nM (¹²⁵I)ANB-NOS-peptide and10 μl of microsomes (60 A₂₈₀/ml) in a total volume of 100 μl RM buffer(250 mM sucrose, 50 mM TEA-HCl, 50 mM KOAc, 2 mM MgOAc₂, and 1 mM DTT).After mixing, the samples were immediately irradiated at 366 nm for 5min at room temperature. The membranes were then recovered bycentrifugation through a 0.5-M sucrose cushion in RM buffer. Themembranes were washed once with cold RM buffer. The washing membraneswere lysed for immunoprecipitation or for immune blotting. Thecrosslinking reaction with ATP contained an ATP regeneration system,described in Li et al (J Biol Chem. 274 (13), 8649-54 (1999)). Thecrosslinking of surface Kb molecules on RAW309Cr.1 cells was performedas mixing 100 nM (¹²⁵I)ANB-NOS-peptide with 10⁷ cells, equivalent toamount of cells used for making 10 μl of microsomes in a total volume of100 μl RM buffer. After mixing, the samples were immediately irradiatedfor 5 min at room temperature. The excess peptides were removed bywashing with RM buffer. The cells were lysed for immunoprecipitationwith Y3 antibody.

The detection of surface MHC class I was performed by incubatingRAW309Cr.1 cells with Y3 antibody at 4° C. for 15 min. After washing,the cells were lysed in 1% NP40 lysis buffer and the cleared lysateswere precipitated with protein-A beads. The precipitated MHC class Iwere detected by immunoblotting with R218 antiserum.

The peptide-editing for stimulation of T cells was performed by mixingmicrosomes with native peptides with ATP regeneration system for 10 minat room temperature.

The excess of peptides was removed after centrifugation through sucrosecushion in RM buffer. The loaded microsomes were repeatedly freeze/thawalternately in liquid nitrogen and then in a water bath at 37° C., for10 times. The processed microsomes were resuspended in PBS atconcentration of (6 A₂₈₀/ml) and kept at −80° C. until use. Thepeptide-pulsed RAW309Cr.1 cells was prepared as mixing peptides 100 nMwith 10⁷ cells in 1 ml medium over night or in 1 ml PBS for four hoursat 37° C. The pulsed cells were either washed with PBS before mixingwith B3Z T cells or add the mixture directly to the B3Z.

EXAMPLE 2 Activation of B3Z T Cell Hybridoma

The prepared stimuli including peptide-edited microsomes, peptide-pulsedRAW309Cr.1 cells, OVA peptide, were added to culture of 10⁵ B3Z cells ina total of 200 μl. Addition of PBS and anti-CD3/CD28 coated beads servedas negative or positive control, respectively. After over nightincubation, the activation of B3Z was represented by LacZ activity usingo-nitrophenyl b-D-galactopyranoside (Sigma) substrate. The linear rangeof OVA-response was determined by the addition of serial dilutions ofSIINFEKL to the medium due to that the B3Z cells themselves express Kband present SIINFEKL.

EXAMPLE 3 Detection of Peptide-Receptive MHC Class I Molecules in theMicrosome, but not on the Surface of APCs

An in vitro peptide transport and loading assay by using crosslinkermodified peptides and isolated microsomes of the ER from RAW309Cr.1 hasbeen reported (Li et al J Biol Chem. 274 (13), 8649-54 (1999)). Theassay allows the examination of both the peptide translocation acrossthe membrane of the ER in the presence of ATP and subsequently thepeptide loading on MHC class I molecules (Wang et al J Immunol. 157 (1),213-20 (1996)).

To detect the peptide-receptive MHC class I molecules in the microsomalmembranes, a crosslinker (ANB-NOS) was conjugated to the ε-amino groupof the lysine residue of an H2-Kb-binding ovalbumin (OVA) peptide(residues 257-264, SIIFEKL) and substituted the isoleucine at position 3with tyrosine to allow for iodination. These modifications allowedphoto-cross-linking of the OVA peptide to H2-Kb molecules during theassembly. For a quantitative comparison of peptide-receptive H2-Kb inmicrosomes and on cell surface of living RAW309Cr.1, the modified OVApeptide was labelled by ¹²⁵I and incubated with microsomes of RAW309Cr.1and living RAW309Cr.1 cells under UV irradiation. Peptide-bound H2-Kbmolecules were subsequently analysed by immunoprecipitation with ananti-H2 antibody Y3. In the absence of ATP, only a few Kb molecules wereassembled with OVA peptides, while a significant amount of Kb moleculeswere cross-linked with OVA peptide in the presence of ATP (FIG. 1). Thisresult confirms that a substantial amount of peptide receptive class Imolecules exist in the ER.

A semi-quantitative analysis of OVA-crosslinked Kb in microsomes and onthe surface of RAW309Cr.1 showed that in contrast to the high levels ofpeptide receptive Kb molecules in the microsomes, the OVA-bound Kbmolecules on the surface of APCs was under the radio-chemical detectivelevel (FIG. 2), suggesting again that peptide-receptive MHC class Imolecules are mainly in the ER, but not on the surface of APCs. In acompeting experiment, it has been shown that the binding of thismodified OVA peptide to Kb is specific. In order to examine the affinityof the modified OVA-peptide, the labelled OVA peptide was competed byits native form at different concentrations. The native OVA peptidecompeted 50% of the report peptide at the concentration of reportpeptides and completely abolished binding at concentration of ten timesof the report peptides (FIG. 2). Moreover, a Ld specific peptide couldnot compete the OVA binding. This shows that the binding affinity ofmodified OVA-peptide is Kb specific and similar to its native form. Toquantitate the amount of peptides bound to Kb molecules in microsomesderived from 10⁶ RAW309Cr.1, the labelled peptides were incubated withmicrosomes in the presence of ATP. After crosslinking, MHC class I wereprecipitated and dpm of peptide-bound Kb was measured and converted tothe concentration of peptides. Results showed that about 500 to 1000peptides were bound to Kb molecules in the microsomes of one cells. Inaddition, the amount of total MHC class I molecules in the ER are morethan that on the surface of RAW309Cr.1 cells (FIG. 3). Thus, microsomesfrom APCs could be able to deliver sufficient peptide-MHC class Icomplexes to T cells.

EXAMPLE 4 B7 and ICAM1 are Presented in the Microsomes of APCs

A full T cell response requires signals from both antigen-MHC complexand co-stimulatory molecules such as B7 (Acuto et al, Immunol Rev. 192,21-31 (2003)). Like all the membrane proteins, co-stimulatory moleculesare synthesised in the ER and subsequently expressed on the surface ofAPCs. To quantitate the amount of co-stimulatory molecules of B7 andICAM-1 in the isolated microsomes, the microsomes equivalent to 5×10⁶RAW309Cr.1 were lysed and the clear lysates were analysed by westernblotting with anti-sera specific to these molecules, respectively. Incomparison, a total cell lysates of 5×10⁶ RAW309Cr.1 were also blottedwith same antibody. The intensity of B7.1, B7.2 and ICAM-1 bands wasquantitated by density analysis. Both B7 and ICAM-1 were readilydetected in the microsomal samples (FIG. 4). The amount detected inmicrosomes was about half of the total cellular lysates. The presence ofsufficient amount of co-stimulatory molecules in peptide-editedmicrosomes could mimic the functional surface of APCs for providing bothantigen-MHC and co-stimulatory signals to T cells.

EXAMPLE 5 Microsomes Loaded With Kb-Specific OVA Peptides Stimulate TCells In Vitro

To investigate the ability for peptide-loaded microsomes to inducespecific T cell response, the native OVA peptide-edited microsomes wereprocessed for inside-out by repeated freeze-thaw method (materials andmethods). The processed microsomes and OVA-peptide pulsed RAW309Cr.1were used to stimulate B3Z T cell hybridoma which recognises Kb-SIINFEKLcomplex (Fremont et al Proc Natl Acad Sci USA. 92 (7), 2479-83 (1995);Shastri N, & Gonzalez F., J Immunol. 150 (7), 2724-36 (1993)). Afterwashing off the excessive peptide, OVA edited Microsome stimulated B3Z Tcells by inducing IL-2 production and the expression of IL-2-promoterdriven LacZ (FIG. 5). The specificity of OVA-Kb induced B3Z responseswas supported by the unresponsiveness of B3Z cells to the microsomeswithout the peptide or loaded with Ld specific peptide (FIG. 5).Moreover, the levels of responses of B3Z to OVA-edited microsomes wascorrelated with the amount of OVA-peptides (FIG. 6). OVA-pulsedRAW309Cr.1 could induce the B3Z response in the presence of excessivepeptides (Schott et al Proc Natl Acad Sci USA. 99 (21), 13735-40(2002)).

However, if excess peptides were removed by washing, theOVA-pulsed-RAW309Cr.1 could no longer induce B3Z response. Given thatOVA itself could induce IL-2 production by B3Z cells (FIG. 5), suggeststhat not RAW309Cr.1, but OVA itself is the stimuli for B3Z. The abilityof SIINFEKL to induce Kb restricted T cell responses in vitro has beenreported recently, suggesting that CTL could present peptides to eachother (Schott et al Proc Natl Acad Sci USA. 99 (21), 13735-40 (2002)).However, the induction CTL response in vitro by peptide-editedmicrosomes, but not by peptide-pulsed RAW309Cr.1 is consistent with thepeptide-binding results (FIG. 2) and indicate that peptide-editedmicrosomes could mimic APCs to efficiently present antigenic peptides toTCR and stimulate full responses of CTLs.

EXAMPLE 6 Microsomes Loaded With Kb-Specific OVA Peptides InducesOVA-Peptide Responses In Vivo

To further examine the ability of OVA-edited microsomes to induce immuneresponses in vivo, OVA-edited microsomes from RAW309Cr.1 cells,microsomes loaded with Ld-specific peptide, soluble OVA peptides,RAW309Cr.1 pulsed with OVA peptides and PBS were used to induce immuneresponse in vivo. Five groups of C57BL/6 or Balb/c mice, each groupconsisting of five mice, were injected twice subcutaneously with abovestimuli, respectively. The interval between injections was one week. Sixdays after second injection, T cells were isolated from spleens andcross-stimulated in vitro with the five original stimuli, respectively.In addition, anti-CD3/CD28 coated beads were used as positive control.PBS stimulated T cells did not respond to any stimulation, whileanti-CD3/CD28 induced proliferative responses in all the groups.OVA-peptide pulsed RAW309Cr.1 and microsomes loaded with Ld-specificpeptide did not induce T cells responses (FIG. 7). In contrast, T cellsfrom C57BL/6 groups of OVA-edited microsomes and OVA peptide respondedto OVA-edited microsomes in vitro, but not to the OVA-pulsed RAW309Cr.1or the microsomes loaded with Ld-peptides (FIG. 7). Compelling resultsfrom IL-2 production (FIG. 7) again support that OVA-edited microsomescould induce specific T cells responses in vivo (FIG. 7). Balb/c hasH-2d, therefore, there was not OVA response induced.

EXAMPLE 7 TCR Signalling Pathways are Activated by OV-Microsomes

In order to analyse the TCR signalling in response to OVA-microsomestimulation, T cells isolated from C57BL/6 mice immunised byOVA-microsomes were used to induce TCR signalling in vitro. Theactivation of ERK and JNK was detected in the T cells stimulated witheither anti-CD3/CD28 or with OVA-edited microsomes, but not withOVA-pulsed RAW309Cr.1 (FIG. 8). Thus, the biochemical evidence indicatesa specific TCR signalling in response to OVA-Kb on microsomes andfurther supports that microsomes edited with antigenic peptides couldinduce specific immune responses in vivo.

EXAMPLE 8 Influenza Viral Peptide Loaded Microsomes

The Kb specific peptide (ASNENMETM) form mouse influenza virus wasloaded into microsomes from Kb specific RAW cells. The loaded microsomeswere used to immunize C57BL/6 mice. In separate groups, the PBS orpeptide were used as controls. After two antigen administrations over aseven day period, T-cells were isolated from spleens and cross-culturedwith PBS or peptide, peptide, or peptide-loaded microsomes for threedays. T-cell proliferation was measured on day 4 after immunization.Results are shown in FIG. 10.

EXAMPLE 9 Effect on Melanoma Cells

T-cells isolated from three A2 melanoma patients (P1, P2, P3),respectively, were stimulated with autologous tumor cells purified fromsurgical biopsies at one to one and with r-human IL-2 (10 U/mL) for fourtimes with a 5 day interval between each administration. The specificanti-tumor responses were tested in comparison to the tumor cell lineK259 by cytotoxic assay. The MAGE peptide was loaded to microsomesisolated from 221-A2 human B-cell line, in which the MHC locus isdeleted, and subsequently transfected with HLA-A2. The T-cell lines frommelanoma patients were cultured with peptide, microsomes, orpeptide-loaded microsomes in normal medium for three days. Theproliferation was measured at day 3. The results are shown in FIG. 12.

DISCUSSION

These results demonstrate that the microsomes derived from the ER can beused to process edited antigenic peptides on MHC class I molecules andthat the processed microsomes can reconstitute the functional surface ofAPCs to induce CTL responses. Thus, the antigenic peptide delivered bymicrosomal MHC class I in association with co-stimulatory molecules is anovel form of peptide vaccine.

REFERENCES TO TABLES 1 TO 7

1. Aarnoudse et al, Int J Cancer 82: 442 (1999)

2. Anichini et al, J Exp Med 177: 989 (1993)

3. Bakker et al, Int J Cancer 62: 97 (1995)

4. Baurain et al, J Immunol 164: 6057 (2000)

5. Bocchia et al, Blood 87: 3587 (1996)

6. Boel et al, Immunity 2: 167 (1995)

7. Bohm et al, Int J Cancer 75: 688 (1998)

8. Boon et al, J Exp Med 183: 725 (1996)

9. Brandle et al, J Exp Med 183: 2501 (1996)

10. Brichard et al, Eur J Immunol 26: 224 (1996)

11. Brossart et al, Blood 93: 4309 (1999)

12. Butterfield et al, Cancer Res 59: 3134(1999)

13. Buzyn et al, Eur J Immunol 27: 2066 (1997)

14. Castelli et al, J Exp Med 181: 363 (1995)

15. Castelli et al, J Immunol 162: 1739 (1999)

16. Chaux et al, J Immunol 163: 2928 (1999a)

17. Chaux et al, J Exp Med 189: 767 (1999b)

18. Chen et al, Proc Natl Acad Sci USA 94: 1914 (1997)

19. Chiari et al, Cancer Res 59: 5785 (1999)

20. Corman et al, Clin Exp Immunol 114: 166 (1998)

21. Correale et al, J Natl Cancer Inst 89: 293 (1997)

22. Coulie et al, J Exp Med 180: 35 (1994)

23. Coulie et al, Proc Natl Acad Sci USA 92: 7976 (1995)

24. Cox et al, Science 264: 716 (1994)

25. Dabovic et al, Mamm Genome 6: 571 (1995)

26. De Backer et al, Cancer Res 59: 3157 (1999)

27. De Plaen et al, Immunogenetics 40: 360 (1994)

28. Dermime et al, Clin Cancer Res 2: 593 (1996)

29. De Smet et al, Immunogenetics 19: 121 (1994)

30. De Smet et al, Mol Cell Biol 19: 7327 (1994)

31. Domenech et al, J Immunol 155: 4766 (1995)

32. Dudley et al, J Exp Med 184: 441 (1996)

33. Duffour et al, Eur J Immunol 29: 3329 (1999)

34. Fisk et al, J Exp Med 181: 2109 (1995)

35. Fleischhauer et al, Int J Cancer 68: 622 (1996)

36. Fleischhauer et al, Cancer Res 58: 2969 (1998)

37. Fujie et al, Int J Cancer 80: 169 (1999)

38. Gambacorti-Passerini et al, Blood 81: 1369 (1993)

39. Gaudin et al, J Immunol 162: 1730 (1999)

40. Gaugler et al, J Exp Med 179: 921 (1994)

41. Gaugler et al, Immunogenetics 44: 323 (1996)

42. Gomi et al, J Immunol 163: 4994 (1999)

43. Greco et al, Leukemia 10: 693 (1996)

44. Gueguen et al, J Immunol 160: 6188 (1998)

45. Guilloux et al, J Exp Med 183: 1173 (1996)

46. Gure et al, Int J Cancer 85: 726 (2000)

47. Heidecker et al, J Immunol 164: 6041 (2000)

48. Herman et al, Immunogenetics 43: 377 (1996)

49. Hiltbold et al, Cancer Res 58: 5066 (1998)

50. Hogan et al, Cancer Res 58: 5144(1998)

51. Hohnet al, J Immunol 163: 5715 (1999)

52. Huang et al, J Immunol 162: 6849 (1999)

53. Ikeda et al, Immunity 6: 199 (1997)

54. Jager et al, J Exp Med 187: 265 (1998)

55. Jager et al, J Exp Med 191: 625 (2000)

56. Jassim et al, Eur J Immunol 19: 1215 (1989)

57. Kang et al, J Immunol 155: 1343 (1995)

58. Kawakami et al, Proc Natl Acad Sci USA 91: 3515 (1994a)

59. Kawakami et al, Proc Natl Acad Sci USA 91: 6458 (1994b)

60. Kawakami et al, J Exp Med 180: 347 (1994c)

61. Kawakami et al, J Immunol 154: 3961 (1995)

62. Kawakami et al, J Immunol 161: 6985 (1998)

63. Kawakami et al, Immunol Res 16: 313(1997)

64. Kawano et al, Cancer Res 60: 3550 (2000)

65. Kawashima et al, Int J Cancer 78: 518 (1998)

66. Kawashima et al, Cancer Res 59: 431 (1999)

67. Kikuchi et al, Int J Cancer 81: 459 (1999)

68. Kittlesen et al, J Immunol 160: 2099 (1998)

69. Kobayashi et al, Cancer Res 58: 296 (1998a)

70. Kobayashi et al, Immunogenetics 47: 398 (1998b)

71. Kono et al, Int J Cancer 78: 202 (1998)

72. Lethe et al, Int J Cancer 76: 903 (1998)

73. Li et al, Cancer Immunol Immunother 47: 32 (1998)

74. Lucas et al, Cancer Res 59: 4100 (1999)

75. Lucas et al, Int J Cancer 87: 55 (2000)

76. Lupetti et al, J Exp Med 188: 1005 (1998)

77. Lurquin et al, Genomics 46: 397 (1997)

78. Mandruzzato et al, J Exp Med 186: 785 (1997)

79. Manici et al, J Exp Med 189: 871 (1999)

80. Martelange et al, Cancer Res 60: 3848 (2000)

81. Minev et al, Proc Natl Acad Sci USA 97: 4796 (2000)

82. Moreau-Aubry et al, J Exp Med 191: 1617 (2000)

83. Morel et al, Immunity 12: 107 (2000)

84. Morioka et al, Mol Immunol 32: 573 (1995)

85. Nakao et al, J Immunol 164: 2565 (2000)

86. Noppen et al, Int J Cancer 87: 241 (2000)

87. Norbury et al, Br J Haematol 109: 616 (2000)

88. Ohminami et al, Blood 93: 925 (1999)

89. Oiso et al, Int J Cancer 81: 387 (1999)

90. Oka et al, Immunogenetics 51: 99 (2000)

91. Panelli et al, J Immunol 164: 4382 (2000)

92. Parkhurst et al, Cancer Res 58: 4895 (1998)

93. Pawelec et al, Blood 88: 2118 (1996)

94. Peiper et al, Eur J Immunol 27: 1115 (1997)

95. Peoples et al, Proc Natl Acad Sci USA 92: 432 (1995)

96. Pieper et al, J Exp Med 189: 757 (1999)

97. Robbins et al, J Immunol 154: 5944 (1995)

98. Robbins et al, J Exp Med 183: 1185 (1996)

99. Robbins et al, J Immunol 159: 303 (1997)

100. Robbins et al, Harwood Acad Publ, London, in press (2000)

101. Rongcun et al, J Immunol 163: 1037 (1999)

102. Ronsin et al, J Immunol 163: 483 (1999)

103. Russo et al, Proc Natl Acad Sci USA 97: 2185 (2000)

104. Salazar-Onfray et al, Cancer Res 57: 4348 (1997)

105. Scanlan et al, Cancer Lett 150: 155 (2000)

106. Schneider et al, Int J Cancer 75: 451 (1998)

107. Shichijo et al, J Exp Med 187: 277 (1998)

108. Skipper et al, J Immunol 157: 5027 (1996)

109. Suzuki et al, J Immunol 163: 2783 (1999)

110. Tahara et al, Clin Cancer Res 5: 2236 (1999)

111. Tanaka et al, Cancer Res 57: 4465 (1997)

112. Tanaka et al, Br J Haematol 109: 435 (2000)

113. Tanzarella et al, Cancer Res 59: 2668 (1999)

114. ten Bosch et al, Leukemia 9: 1344 (1995)

115. ten Bosch et al, Blood 88: 3522 (1996)

116. ten Bosch et al, Blood 94: 1038 (1999)

117. Topalian et al, Proc Natl Acad Sci USA 91: 9461 (1994)

118. Topalian et al, J Exp Med 183: 1965 (1996)

119. Traversari et al, J Exp Med 176: 1453 (1992)

120. Tsai et al, J Immunol 158: 1796 (1997)

121. Tsang et al, J Natl Cancer Inst 87: 982 (1995)

122. van Baren et al, Br J Haematol 102: 1376 (1998)

123. Van den Eynde et al, J Exp Med 182: 689 (1995)

124. Van den Eynde et al, J Exp Med 190: 1793 (1999)

125. van der Bruggen et al, Science 254: 1643 (1991)

126. van der Bruggen et al, Eur J Immunol 24: 3038 (1994a)

127. van der Bruggen et al, Eur J Immunol 24: 2134 (1994b)

128. Visseren et al, Int J Cancer 73: 125 (1997)

129. Vissers et al, Cancer Res 59: 5554 (1999)

130. Vonderheide et al, Immunity 10: 673 (1999)

131. Wang et al, J Exp Med 184: 2207 (1996a)

132. Wang et al, J Exp Med 183: 1131 (1996b)

133. Wang et al, J Immunol 160: 890 (1998a)

134. Wang et al, J Immunol 161: 3598 (1998b)

135. Wang et al, Science 284: 1351 (1999a)

136. Wang et al, J Exp Med 189: 1659 (1999b)

137. Wolfel et al, Eur J Immunol 24: 759 (1994)

138. Wolfel et al, Science 269: 1281 (1995)

139. Yang et al, Cancer Res 59: 4056 (1999)

140. Yasukawa et al, Blood 92: 3355 (1998)

141. Yotnda et al, J Clin Invest 101: 2290 (1998a)

142. Yotnda et al, J Clin Invest 102: 455 (1998b)

143. Yun et al, Tissue Antigens 54: 153 (1999)

144. Zarour et al, Proc Natl Acad Sci USA 97: 400 (2000)

145. Zeng et al, J Immunol 165: 1153 (2000)

146. Zorn et al, Eur J Immunol 29: 592 (1999a)

147. Zorn et al, Eur J Immunol 29: 602 (1999b)

1-31. (canceled)
 32. A vaccine composition comprising an isolated inverted microsome from an animal cell, or a membrane fragment thereof, in association with an externally disposed peptide antigen and a protein of the Major Histocompatibility Complex (MHC).
 33. A composition according to claim 32 in which the microsome is from the endoplasmic reticulum of the cell.
 34. A composition according to claim 32 in which the protein of the MHC is from a heterologous source with respect to the cell from which the microsome is obtained.
 35. A composition according to claim 32 in which the composition additionally comprises one or more co-stimulatory molecules.
 36. A composition according to claim 35 in which the co-stimulatory molecule is B7 or IL-2.
 37. A composition according to claim 32 in which the antigen is from a viral, bacterial, yeast, fungal, or protozoan origin.
 38. A composition according to claim 32 in which the antigen is an auto-antigen.
 39. A composition according to claim 37 in which the antigen is of neoplastic cell or cell of a cancer tumour, or a normal self-protein.
 40. A composition according to claim 39 in which the neoplastic cell or cancer cell tumour is from a melanoma, lung adenocarcinoma, colon cancer, breast cancer, or leukemia cell.
 41. A kit of parts comprising a composition according to claim 32 and one or more cytokines and/or adjuvants in sealed containers.
 42. A kit of parts according to claim 41 in which the cytokine is IL-2 or IFNγ.
 43. A kit of parts comprising a composition according to claim 32 and one or more cytokines and/or adjuvants for separate, subsequent or simultaneous administration to a subject.
 44. A kit of parts according to claim 43 in which the cytokine is IL-2 or IFNγ.
 45. A process for the preparation of a vaccine composition according to claim 32 comprising incubating a population of microsomes and an antigen in the presence of a nucleoside triphosphate, followed by processing to prepare inverted microsomes, and formulating the resulting preparation in a physiological diluent and optionally an adjuvant.
 46. A method of treatment or prophylaxis of a subject suffering from a disease or condition, comprising administering to the subject a vaccine composition according to claim 32 to treat said disease or condition. 